Blood platelets are one of the complex components involved in maintaining hemostasis. When a blood vessel wall is damaged, platelets adhere to exposed surfaces composed of collagen, microfibrils, and basement membrane. Adherent platelets promote the recruitment of other platelets to form an aggregated mass called a hemostatic platelet plug. The result is to activate coagulation proteins which provide the network to stabilize the platelet plug and reduce bleeding, allowing tissue repairs to occur.
Platelets are transfused to patients for many clinical indications. For instance, platelet infusions are used to correct deficiencies or dysfunctions of circulating platelets as a result of trauma, disease, or drug induced dysfunction. Patients suffering from idiopathic thrombocytopenia and those undergoing ablative chemotherapy are treated with platelet infusions. The increasing use of ablative chemotherapy for a wide variety of malignancies has resulted in an increased need for replacement platelet therapy.
A major difficulty in using isolated platelets is their short shelf-life. Platelets are only approved by the Food and Drug Administration (FDA) for storage in a liquid state for up to five days at room temperature, during which time the functional properties rapidly deteriorate. This causes many logistic problems in both civilian and military medicine.
Further drawbacks of storing platelets in a liquid state include the necessity of considerable storage space and constant agitation within bags of specially developed gas permeable plastics. Typically, platelets are stored in a suspending plasma volume of 45 to 65 mL. Recently, a study reported liquid storage establishing a minimum plasma volume of 30-50 mL. Home et al. (1994) Transfusion 34:39; and Ali et al. (1994) Transfusion 34:44. This storage method still requires considerable space, however, and the shelf life is not extended beyond approximately five days. The major problem with liquid storage is that the platelets need to be stored above 20.degree. C. as even short periods of exposure to lower temperatures during storage result in substantial changes in their in vivo and in vitro properties. Moroff et al. (1994) Transfusion 34:317. As this storage usually also requires agitation of the platelets during storage at about 20-24.degree..degree.C., often in the presence of glucose, this presents optimal conditions for bacterial growth which is a major problem with the storage of liquid platelets.
To minimize the problems of bacterial growth, refrigerated storage at 4.degree. C. or frozen storage at -80.degree. C. has been proposed. However, this requires methods to prevent the cold-activation of the stored platelets. The use of anti-freeze glycoproteins have also been suggested for use in preservation of platelets. Tablin et al. (1996) in Frozen Platelets and Platelet Substitutes in Transfusion Medicine, Uniformed Services University of the Health Sciences (the "Bethesda meeting"). This method would however, require extensive washing of the platelets, to remove the anti-freeze proteins, before therapeutic use. This drawback is also seen with the methods of freezing platelets involving the use of DMSO which would again require extensive washing of the platelets before therapeutic use. Bock et al. (1995) Transfusion 53:921. A method of storing platelets by freeze drying has been described. Bateson et al. (1994) Transfusion Med. 4:213, EPA 0356257. Platelets are activated during the process of freeze drying and can only be used as a hematology standard and the electrokinetic properties of the preserved platelets are different from those of fresh matched platelets. Oliver et al. the Bethesda Meeting. Other attempts at lyophilizing platelets have met with suboptimal results. Bode (1993a and b) First and Second Triannual Reports, (respectively) Evaluation of Dried Storage of Platelets for Transfusion: Physiologic Integrity and Hemostatic Functionality. DNR Grant No. N00014-92-J-1244. Fixing the platelets prior to freeze-drying improves their function but these freeze-dried platelets need to be stored frozen at -80.degree. C. Read et al. (1995) Proc. Natl. Acad. Sci. 92:397.
At the Bethesda meeting, the following ideal attributes for platelets were stated: prevention of bleeding for up to 24 hours; 20% of infused platelets in circulation after 24 hours; long-term storage outside of freezers or refrigerators; lightweight, durable and easy to transport; easy to use without the need of centrifugation; non-immunogenic; sterile; and donor check possible after three months. The meeting also highlighted the lack of in vivo models for the evaluation of, and data on the in vivo evaluation of, most human platelet preparations being developed. One exception was the reticulo-endothelial-system-block thrombocytopenic rabbit model of Blajchman (RES-block model) which enabled the evaluation of human platelet efficacy in an animal model. Ali et al. (1994) Transfusion 34:44; and Blajchman et al. (1994) Brit. J. Haematol. 86:347.
Platelet products have also been proposed for use as platelet substitutes and as independently therapeutic agents. These products also offer a longer shelf life than intact platelets. Platelet membrane vesicles have been prepared by a number of methods and have been shown to reduce bleeding time in a rabbit model. Chao et al. (1996) Transfusion 36:536-542; and U.S. Pat. Nos. 5,428,008 and 5,332,578. Dried, powdered platelet membranes have also been prepared as an immunoadsorbent. EP 141939.
It would be useful to fulfill many of the goals enumerated above. It would be especially useful to have a method for storing platelets at ambient temperatures for periods of time greater than five days. These criteria immediately eliminate the applicability of all current liquid and cryopreserved platelet preparations. Even more advantageous would be a method for long term storage of dried platelets, particularly with regard to the problem of bacterial growth during liquid storage. Storage of dried platelets would require less space than storage of platelets in liquid or as a frozen solid due to reduced volume and would not require constant agitation in the liquid state, thus enabling considerable savings on storage and transport costs. This would also enable the use of platelets in areas of the world that do not otherwise have adequate storage or transport facilities. Banking of HLA-matched dried platelets would also find use in treatment of alloimmunized patients.
Trehalose, .omega.-D-glucopyranosyl-.omega.-D-glucopyranoside, is a naturally occurring, non-reducing disaccharide which was initially found to be associated with the prevention of desiccation damage in certain plants and animals which can dry out without damage and revive when rehydrated. Trehalose has been shown to be useful in preventing denaturation of proteins, viruses and foodstuffs during desiccation and subsequent storage. See U.S. Pat. Nos. 4,891,319; 5,149,653; 5,026,566; Blakeley et al. (1990) Lancet 336:854; Roser (July 1991) Trends in Food Sci. and Tech., pp. 166-169; Colaco et al. (1992) Biotechnol. Internat., pp. 345-350; Roser (1991) BioPharm. 4:47; Colaco et al. (1992) Bio/Tech. 10:1007; and Roser et al. (May 1993) New Scientist, pp. 25-28.
In nature, trehalose stabilizes the cell membrane under various stressful conditions. Trehalose is linked to the ability of yeast cells to survive complete dehydration. Eleutherio et al. (1993) Biochim Biophys Acta 1156:263. Trehalose is also known to stabilize lyophilized proteins, such as methanol dehydrogenase (Argall and Smith (1993) Biochem. Mol. Biol. Int. 30:491), and to confer thermoprotection to enzymes from yeast. Hottiger et al. (1994) Eur. J. Biochem. 219:187.
Trehalose has been used to stabilize lyophilized sarcoplasmic reticulum and liposomes. Crowe, J. et al. (1983) Arch. Biochem. Biophys. 220:470-484; and Crowe, L. et al. (1984) Biochim. Biophys. Acta 769:141-150. In both these instances, trehalose was added to the solution in which the membranes or liposomes were suspended, the mixture was frozen and freeze dried. Trehalose was found to prevent membrane phase transition. Trehalose has been suggested for use in the cryopreservation of platelets; the loading of these platelets by freeze-induced membrane phase transition has been reported to prevent cold-induced activation during freezing. Oliver et al. the Bethesda Meeting. However, these platelets need to be stored frozen at -80.degree. C. and the use of DMSO for the cryopreservation would require the platelets to be washed extensively before therapeutic use Oliver et al. and Crowe, L. et al. (1996) the Bethesda Meeting. Although a number of carbohydrates have been suggested for use in lyophilizing red blood cells, both trehalose and sucrose were found to be unsuitable. EP 356,257.
The methods described herein provide methods for preserving platelets by drying and the compositions obtained thereby. The dried platelets can be stored indefinitely at ambient temperatures. During storage the functional properties of the platelets are unchanged.
All references cited herein, both supra and infra, are hereby incorporated herein by reference.